The recently described type VII secretion system, exemplified by the archetypal ESX-1 apparatus in Mycobacterium tuberculosis, is a key virulence determinant in many Gram-positive bacteria. ESX-1 is a highly conserved apparatus, which secretes a specific set of proteins that promote virulence by modulating the immune response and helping to disseminate the bacteria through the host. M. tuberculosis ESX-1 mutants are attenuated. Despite the importance of this apparatus, little is known about the structural proteins, how, or where they are assembled and the mechanism of secretion. This proposal will address these deficits. This laboratory has defined a role of ESX-1 secretion in modulating conjugal DNA transfer in the model mycobacterium M. smegmatis. All of our genetic and biochemical studies indicate that the ESX- 1 apparatus and its secreted substrates are functionally interchangeable between M. smegmatis and M. tuberculosis;M. smegmatis will secrete M. tuberculosis ESX substrates, and M. tuberculosis esx-1 genes will complement M. smegmatis ESX-1 mutants. Moreover, the fast-growth rate, its genetic tractability and the non-pathogenic nature of M. smegmatis make it an ideal system to dissect the ESX- 1 apparatus. We will build upon preliminary data that suggest the ESX-1 apparatus localizes to the cell poles. Using a combination of fluorescence microscopy and molecular genetics we will define those proteins assembled into the core machinery, their mechanism of assembly, and look for small molecule inhibitors of secretion. The specific aims are to: 1. Identify the cellular location of ESX-1 associated proteins in M. smegmatis. 2. Determine the order of assembly of ESX-1 and the proteins mediating this process. 3. Develop a simple fluorescence-based assay to monitor ESX-1 secretion. The similarities of type VII secretion systems among Gram-positive bacteria will ensure that models developed from this work will be broadly applicable to other bacterial pathogens including B. anthracis and C. diphtheriae. Most importantly, the recent emergence of extremely-drug resistant strains of M. tuberculosis has increased the demand for new treatments for a disease that kills over 1.6 million people a year: the ESX-1 apparatus represents a potential new target for rational drug design. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis accounts for over 1.6 million deaths per year. In order to define rational new drug targets to combat this organism, there is a need to understand its biology and how it survives within host cells. M. tuberculosis secretes proteins that are known to modulate the host response. The goal of this work is to identify proteins required for this secretion process and how they assemble to form a secretion machine. As secretion is critical for virulence we anticipate that the apparatus will provide a new drug target.